Comparative Antioxidant Content and Antioxidant Activity

of Selected Romanian and Polish Honeydew Honey

ADRIANA MONICA CHIS1*, CORNELIA PURCAREA1, MALGORZATA DZUGAN2, ALIN TEUSDEA3
1
University of Oradea, Environmental Protection Faculty, Department of Food Industry, 26 Gen. Magheru Str, 410048, Oradea,
Romania
2
University of Rzeszow, Faculty of Biology and Agriculture, Poland
3
University of Oradea, Environmental Protection Faculty, Department of Animal Science-Agritourism, 26 Gen. Magheru Str.,
410048, Oradea, Romania

The aim of this study was to investigate and to compare the main antioxidant components and the radical
scavenging activity (RSA) of honeydew honey from selected counties in Poland (Podkarpackie) and Romania
(Bihor). Total phenols (TPH), flavonoids (FL) and ascorbic acid (Vitamin C, VC) content were determined as
antioxidant components. For RSA the DPPH method was applied on honey solutions at different
concentrations in order to determine the IC50 value and antioxidant activity of 1% honey solutions (AA1%).
The results show high antioxidant content for all samples: TPH from 107.9 (RO1) to 215.6 (PL3) mg GAE/
100 g, FL from 14.92 (PL4) to 20.36 (PL2) mg QE/100g and VC from 13.02 (RO1) to 16.27 (PL2) mg/100 g.
All samples show very good RSA with IC50 value from 2.39 (PL4) to 5.11 (PL2) % and antioxidant activity of
1% honey solution between 7.04 (P2) and 36.12% (PL3). A strong correlation factor TPH - RSA was found for
both series of samples (>0.824). Only for RHD we found that FL and VC were correlated to AA1%.
Keywords: honeydew honey, phenols, flavonoids, vitamin C, DPPH assay, correlation

Honey, as the European legislation stipulates, is a natural

sweet substance produced by bees from two different
sources. So there is blossom, mono or polifloral honey
obtained from nectar of various plants and honeydew
honey. This last ones source is the secretions of living parts
of plants and the excretions of plant sucking insects [1].
Related to primary component honeydew honey is also
called forest, oak, fir or pine honey. No matter the type, all
honey is seen as an important energetic source for the
organism due to its high sugar content, at least 60% for
honeydew honey. Besides the nutritional role that some
components of this food give it, since ancient times, it was
very important for human health in many directions. The
minor components responsible for the antioxidant activity
of honey are poliphenols (phenolic acids and flavonoides),
vitamins (vitamin C), enzymes (catalase, peroxidase,
glucose-oxidase), amino acids (proline) and carotenoides.
The role of honey polyphenols in health including
antimicrobial, antiviral, antitumor, antiulcer and
cardioprotective properties was reviewed by [2].
It is common knowledge that a lot of vegetal origin
foods have high antioxidant content and activity, especially
red fruits [3-5]. Among animal origin food honey has
a singular position from this point of view. In recent years,
the concerns about human health in relation with nutrition
led to scientific preoccupations on honeys antioxidant
content and radical scavenging activity (RSA) all over the
word and very different types of honey seem to have high
poliphenolic content and considerable RSA. This is the case
of acacia ehrenbergina from Yemen [6], Vitellaria honey in
Burkina Faso [7], Millefiori honey from Italy [8], heather
honey from Portugal and Romania [9, 10], manuka honey
from Australia [11]. Considering the forest area in Central
East European countries, the studies on honeydew honey

antioxidant properties is justified, moreover its commercial

value is higher than for the floral types. Most of them
compare honeydew honey to floral honey [12-17],
revealing their higher polyphenol and flavonoid content and
a greater RSA. Other researches refer only to honeydew
honey from different areas in Romania [18] or Slovakia
[19].The comparison between reported results is not
always evident in all cases because some of the studies
refer to entire honey, other to methanolic extracts on
Amberlite. A review concerning the analysis of phenolic
acid and flavonoids in honey was published in 2009 [20]
but there has not been any standardisation in this field of
research activity until now. That is the same for the applied
methods to determined RSA in different matrix [21] or
dedicated especially to honey [22]. The main difference is
that the involved mechanism includes hydrogen atom
transfer (HAT) or electron transfer (ET) reaction-based
methods. DPPH assay used in this study is ET type namely
detect the ability of a potential antioxidant to reduce any
compound including radical [23].
Since the extent of antioxidant content, especially
polyphenols, depend on floral source, geographical and
climatic conditions [2, 24] it is important to investigate
those valuable components even in the same type of honey
but from different regions. The aim of our study was to
quantify the main antioxidant component and to determine
the RSA of selected Romanian and Polish honeydew honey
from distinct geographical areas. In the same time we
have investigated the correlation between those
components and RSA. As far as our knowledge goes, the
above mentioned studies regarding honeydew honey
antioxidant activity already published refer to other counties
as origin for the tested samples; they are Transylvania for
Romania and Pomerania, Malopolska and Bieszczady for
Poland.

* email: andichis@yahoo.com
214

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REV.CHIM.(Bucharest) 67 No.2 2016

Experimental part
Material and methods
Romania - Five samples of honeydew honey (RHD) were
analyzed, three of them from 2012 (fir RO3) and honeydew
(RO1 and RO5) and two from 2013: fir (RO4) and
honeydew (RO2). The samples were purchased from
beekeepers from Bihor County.
Poland - Five samples of honeydew honey (PHD) were
analyzed, one was from 2012 (PL1) and four from 2013.
Two of those last samples ware labelled as ecological
honeydew honey (PL2 and PL3) and two usual honeydew
honey (PL4 and PL5). The samples come directly from
beekeepers from Podkarpackie County).
Antioxidant components
Total phenolic content (TPH) content was determined
by Folin-Cioclteu colorimetric method as described by [7].
From each sample, 500 L of 0.1 mg.mL-1 honey filtered
solution were mixed to 2.5 mL 0.2N Folin-Ciocalteu
Reagent (MERCK Germany). After 5 min, 2 mL of 7.5%
sodium carbonate (Chimopar Romania) solution were
added and mixed in a vortex (HETTIK Germany). The
absorbance was read after 2 hours (room temperature) at
760 nm (spectrophotometer UVMini-1240 Shimatzu)
against a methanolic blank (SCHARLAU Germany). Gallic
acid (ROTH Germany) was used as standard for the
calibration curve from 0 to 250mg.L-. TPH content was
expressed as mg of Gallic acid equivalents (GAE)/100 g of
honey using the calibration curve, y = 0.0094x + 0.0316 ,
R2 = 0.9956 .
Flavonids (FL) content of honey was estimated by
aluminium chloride (AlCl3) colorimetric method [25]. In a
test tube, one ml of each honey solution (0.2 mg.mL-1 in
80% ethanol- SCHARLAU Germany) was reacted with 3
mL of 95% ethanol, 0.2 mL of a 10% aqueous solutions of
AlCl 3 (Alfa Aesar), 0.2 mL of 1 M potassium acetate
(CHEMOPAR Romania) and 5.6 ml of distilled water. The
mixture was shacked for 30s in a vortex and subsequently
allowed to rest at room temperature for 30 min and then
the absorbance was read at 415 nm. For the blank the
AlCl3 solution (0.2 mL) was replaced by distilled water.
Quercetin ((ROTH Germany) was the standard and the
stock solution (250 mg.L-1) was diluted from 0 to 100
mg.L-1 with 80% ethanol applying subsequently the same
steps as for the samples to one ml of each diluted solution.
FL content is expressed as mg of Quercetin equivalents
(QE)/100 g of honey using the calibration curve, y = 0.0059x
0.005, R2 = 0.9933.
Ascorbic acid (vitamin C, VC) content was determined
by the 2,6-dichlorophenolindophenol (MERCK Germany)
colorimetric method [9]. One hundred mg honey sample
was extracted with 10 mL of 1% metaphosphoric acid
(MERCK Germany) for 45 min at room temperature and
filtered. The filtrate (one mL) was mixed with 9 mL of
2,6-dichlorophenolindophenol (MERCK Germany) and the
absorbance was measured within 30 min at 515 nm
against a blank. A solution of pure l- ascorbic acid (ROTH
Germany) from 0.020 to 0.12 mg.mL-1 gave the calibration
curve, (y = 3.102 x - 0.0652; R 2 = 0.9968) and the
results were expressed as mg of ascorbic acid/100g of
honey.
Antioxidant activity
The antioxidant activity was determined by a DPPH
assay (1,1-diphenyl 1,2-picrylhyhrazyl) radical using a
combination of [7, 18, 26] methods with some
modifications. Stock 1 mg.mL-1 DPPH solution (Alfa Aesar)
in methanol was diluted to 0.04 mg.mL-1 for use in the
REV.CHIM.(Bucharest) 67 No.2 2016

assay. From each honey samples aqueous solutions from

10% or 5% (depending of their activity against DPPH
radical) to 0.5% was prepared, filtrated and then
absorbance at 517 nm was determined for colour correction
purposes. The reaction mixtures contain 750 L of each
honey solution and 1500 L of DPPH solution and were
prepared in parallel in 1cm path length plastic cuvettes
stables to the used solvents. The blank contains distilled
water instead of honey solution. Absorption at 517 nm was
determined at increasing period of time and sixty minutes
was the experimental time needed for reaching stable
values [26] for the tested honeydew honey. Only pale pink
solutions were taken into consideration and RSA was
calculated for each of them as %Inhibition = (Ablank Asample)/
Ablank x 100. A graph of honey solution concentration against
%. Inhibition was prepared in order to calculate the IC50%
values and antioxidant activity of 1% honey solution (AA1%).
This study was conducted in the food control laboratory
of the Environmental Protection Faculty, University of
Oradea in 2013-2014.
Statistical analysis
Statistical analyses were performed for two samples in
duplicates and the data was expressed as mean standard
deviations (SD). The means of the determined parameters
of honeydew honey were compared by Kruscal-Wallis for
two independent samples. Differences between means
at 95% (p<0.05) confidence level were considered
statistically significant. Correlations were obtained by
Spearmans correlation coefficient (rs) in bivariate linear
correlations. Associations between the variables were
carried out by bivariate linear regressions. Results with
P<0.05 confidence level were considered statistically
significant. All the statistical analyses were performed with
the PAST version 2.17c software, (Palaeontology Statistics,
Copyright yvind Hammer and D.A.T. Harper (February
2013), http://folk.uio.no/ohammer/past/)(yvind Hammer,
2005).
Results and discussions
Table 1 shows the results obtained for Romanian and
Polish tested honeydew honey samples as mean values
and standard deviation. They refer to the antioxidant
components: total phenols (TPH), flavonoids (FL), ascorbic
acid (VC) and the antioxidant activity expressed as IC50
and AA1%. IC50% is defined as the concentration of honey
causing the decrease of initial DPPH concentration by 50%
and AA1% represents the antioxidant activity of 1% honey
solutions.
For TPH, the lowest content was registered for RO1
(107.9) and the highest for PL3 (215.6). Thus our results
comply with those found in Romania by [18, 27] from 93.5
to 286.6, but surpass those reported in Poland by [16 and
17] from 26.3 to 71.9. These results indicate the fact that
not only the origin of honey affects its phenolic content but
also the geographical area and specific climatic conditions
related to it or to the year of harvest. All compared results
are express in mgGAE/100g.
FL content is significant and ranges between 14.92 (PL4)
and 20.36 (PL2) mg QE/100 g, from far more homogenous
in Romanian honeydew honey samples. Only [13] found
for honeydew values in the same area (28.25), other
studies [7, 18, 25], mentioned lower values, fewer than
5.4; all compared results are expressed as mg QE/100g.
VC content ranges between 13.02 (RO1) to 16.27 (PL2)
mg/100g. These values comply with those found by [9] for
different Portuguese honey (14.58 mg/100g) but the other
cited studies on honeydew honey do not report data about
VC content. As a particular observation, ecological

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215

Table 1
ANTIOXIDANT COMPONENTS AND
ANTIOXIDANT ACTIVITY (DPPH TEST)

honeydew honey sample PL2 shows the greatest content

of flavonoids and vitamin C from all the tested samples.
Statistical analysis between RHD and PHD mean values
(table 1) discloses no significant differences for TPH and
significant differences for FL and VC (p < 0.05). SD values
show a different dispersion of the experimental data for
Romanian respectively Polish honeydew, even if the
number of the tested samples is the same. Romanian
samples show higher homogenous experimental data on
all tested parameters. That could be due to the fact that
two of the Polish samples were ecological and they differ
even as appearance from the others. The usual
honeydew honey samples are dark brown, opaque, viscid
and without signs of cr ystallisation meanwhile the
ecological ones are light brown, opaque and crystallised.
The assumption of being mixed with floral honey having
high crystallisation rate like lime or rape do not seem to be
appropriate because the phenol content is high, for PL3
even the highest of all tested samples. The values for total
phenols in rape and lime honey were always inferior to
those found in honeydew honey [14, 16, 17, 28]; reported
values for phenol in Polish lime honey fall from 15.17 to
47.14 mg GAE/100g and for Polish rape honey from 9.6 to
41.17 mg GAE/100g, which is much lower. The same
situation was observed for Romanian Lime honey [13, 29]:
16.0 to 53 mgGAE/100g
DPPH assay, first reported by [30], is widely used
nowadays for RSA determination in honey due to the fact
that 1,1-diphenyl 1,2-picrylhyhrazyl radical is stable and
easy to use, the procedure is simple and needs only usual
laboratory device such is a UV-Vis spectrophotometer. But
there is a lot of variability regarding especially DPPH and
substrate concentrations, which can be a problem when
the purpose is to compare antioxidant activity of different
216

kinds of honey or the same type but from different

geographical areas. In this conditions the use of IC50 and
AA1%, previously defined, seems to be more appropriate
for comparison reasons at it concern the antioxidant
activity
IC50 values, range from 2.39% (PL4) to 5.11% (PL2).
These values are lower than those reported by [18] for
Romanian honeydew honey, Transylvania Region, (16.16%)
but higher than for Slovenian honeydew honey [12]: 0.82%.
Concerning the AA1% the lowest value 7.04% was recorded
for PL2 and the highest 36.12% for PL3. For 1.25% honey
concentration a bigger inhibition, from 40 % to 61% was
found by [10] and [13]. Honeydew honey has a particular
no floral source but it is not really uniform because the
host of the sucking insects differs: fir, pine, spruce or
oak. So even they are evident particularities for honeydew
honey as dark colour or high phenolic content for example,
some differences regarding the antioxidant activity,
otherwise high, are normal. Statistical analysis (table 1)
shows no significant differences for antioxidant activity of
investigated Romanian and Polish honeydew honey
samples.
Correlation matrix of all five variables was calculated
for each country sample (table 2). The Spearmans (rs)
correlation coefficient was used. The upper diagonal half
part of the matrix represents the statistical significance
(p). Statistical significant (p<0.05) correlations for both
countries were encountered between: TPH, FL and RSA
expressed by IC50 and AA1%. These pairs perform high
values of correlation coefficients (rs>0.700), that
represents a strong correlation between these variables.
For samples from both countries, this indicates that the
involved honey parameters respond in the same way
disregard the honey geographical origin.

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REV.CHIM.(Bucharest) 67 No.2 2016

Table 2
SPEARMANS CORRELATION COEFFICIENT,
rs, BETWEEN HONEY PARAMETERS, WITH
STATISTICAL SIGNIFICANCE

Table 3
BIVARIATE LINEAR REGRESSION
STATISTICAL RESULTS OF THE
DEPENDENT VARIABLES IC50% AND AA1%

These results for TPH-RSA correlation comply with those

reported by [9, 12, 14, 28]: 0.880 to 0.932, but are higher
than those found by [7, 16]: 0.500 to 0.740. This named
study does not refer exclusively to honeydew honey but at
different types of honey, many of them including honeydew
honey. Thus the experiment is validated from this point of
view. The FL variable performs statistical significant
(p<0.05) and high correlation coefficient values with: TPH,
VC and AA1% variables in case of samples from Romania,
fact, in our knowledge, not reported before by other studies.
Associations of analysed honey parameters were
investigated by bivariate linear regressions. Regression
REV.CHIM.(Bucharest) 67 No.2 2016

results for dependent variables: IC50% and AA1%, are

presented in table 3.
These analysis show a negative linear correlation
between TPH and IC50 for honeys from both countries
which comply with the previously cited studies and also a
positive linear correlation between TPH and AA1% for
honeys from both countries, fact not previously reported.
Statistical significant (p<0.05) and high absolute values
of correlation coefficients (R>0.85), from bivariate linear
regressions are displayed only between RSA (IC50 and
AA1%) and TPH.

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217

Conclusions
The study shows that the selected Romanian honeydew
honey (Bihor County) and Polish honeydew honey
(Podkarpatie County) are rich in antioxidant components
such as total phenols, flavonoids and ascorbic acid. They
also have an important radical scavenging activity so they
can be an important source of antioxidants in human diet.
The high correlation observed between TPH and RSA
emphasise the idea that phenols are the main contributor
in antioxidant activity in honeydew honey (more than 85%).
The results that validate the experiment, enables the
hypothesis that variable FL is related (with strong
correlation), as independent variable with TPH, VC and
antioxidant activity.
Folin-Ciocalteu method and DPPH assay standardisation
for specific matrix including honey would be a very
important step in the comparative investigation of
antioxidant capacity in various foodstuffs.
The study discloses no significant differences
concerning the content of total phenols content and radical
scavenging activity between tested honeydew honey even
if in PHD two samples were labelled as ecological and
in RHD two were specified as fir. Thus the primary source
of honeydew honey seems to have low impact upon its
antioxidant activity. Further experiments in both named
counties are needed to extend the present results on more
samples and by alternative antioxidant activity tests too.
Moreover, special mixed preparation containing
honeydew honey may be an interesting subject of study in
the field of functional foods.
Acknowledgments: We are grateful to our colleagues from Rzeszow
University, Faculty of Biology and Agriculture for providing the Polish
honeydew honey samples.

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Manuscript received: 2.02.2015

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